Significance The nef gene of SIV is important for maintaining high virus load and progression to simian AIDS (SAIDS) in adult rhesus macaques. Nef of HIV and SIV contains highly conserved sequence motifs. This sudy is part of an ongoing analysis of functional domains of Nef in vitro (cell-free and tissue culture assay systems) and in vivo (macaques infected with mutant SIV nef clones). Objectives The nef gene of SIV is important for maintaining high virus load and progression to simian AIDS (SAIDS) in adult rhesus macaques. Nef of HIV and SIV contains a highly conserved src-homology region 3 (SH3)-ligand domain characterized by the pro-x-x-pro motif. The objective was to determine the importance of this motif in viral pathogesis by evaluating a mutant clone, in which both pro residues were mutated to ala residues, in vivo in macaques. Results The mutant clone SIVmac239-P104A/P107A, containing mutations in the SH3-ligand domain of Nef, was not able to interact with the Nef-activated kinase (NAK) in in vitro kinase assays. Macaques were inoculated with this mutant to test the in vivo significance of this domain. Genotypic and phenotypic reversions in nef were observed in viruses recovered from these animals. The reversions correlated with viral persistence and progression to SAIDS. Thus, the interaction of Nef with NAK is important for viral pathogenesis. Future Directions The cellular protein(s) interacting with the SH3-ligand domain of Nef remains to be identified. Novel antiviral strategies can be developed based on drugs that interact with the SH3-ligand domain of Nef. KEYWORDS SIV, viral pathogenesis